Electrochemical aptasensors with a gelatin B matrix

ABSTRACT

This invention provides:—an aptamer-based electrochemical sensor, wherein said aptamer is covalently bonded to or chemisorbed on an electrode, said aptamer forming a complex with a target molecule and is encapsulated by a gelatin B matrix;—a method of manufacturing said aptamer-based electrochemical sensor;—the use of the aptamer-based electrochemical sensor for the electrochemical determination of a concentration of a target molecule; and—a composite electrode combining a polymeric material and electrically conducting particles for selective analyte detection, wherein said electrode is coated with gelatin type B.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/901,760 having a 371(c) date of Dec. 29, 2015, which is theU.S. National Stage of International Application No. PCT/EP2014/064249,filed on Jul. 3, 2014, which claims the benefit under 35 U.S.C. § 119(e)of European Patent Application No. 13175128.1, filed Jul. 4, 2013.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to electrochemical aptasensors with agelatin B matrix, a method of manufacturing the same and their use fordetermining target molecule concentrations. The present inventionfurther relates to a composite electrode combining a polymeric materialand electrically conducting particles for selective analyte detection.

BACKGROUND OF THE INVENTION

Electrochemical techniques are recognized as very important candidatesfor the development of biosensors to be used in non-specializedenvironments. Although there has been intensive research intoamperometric biosensors and some of them have successfully reached thecommercialization stage, there have been few reports of the use ofbiomolecules in potentiometric sensors except for the use of antibodiesfor the detection of bacteria, viruses and marker proteins viapotentiometric principles.

De Wael et al. in Analytical Chemistry (2012) 84:4921-4927, reported thefirst use of potentiometric sensors to study molecular interactions inliquid environments with sensorgram methodology.

Aptamers have shown enormous potential in selectively detecting drugs,toxins, proteins etc. Sassolas et al., Electroanalysis 21(11) (2009)1237-1250 reported the use of aptamers as the bio-recognition element inpotentiometic sensors. When combined with Ion Sensitive Field EffectTransistors (ISFETS) as readout systems in poten-tiometric sensors,aptamers appear better than antibodies, due to their smaller size.

Pilehvar et al. in Anal. Chem. (2012) 84:6753-6758, reported a novel,label-free folding induced aptamer-based electrochemical biosensor forthe detection of chloramphenicol (CAP) in the presence of its analogues.

Despite the fact that aptamers are chemically more stable than proteins,they have to be protected from nucleases. Moreover, in respect ofelectrochemical biosensors, the electrode surface also needs to beprotected from unspecific adsorption and oxidation/reduction reactionsoccurring while analyzing real samples.

WO 2005/103664 discloses composite potentiometric electrodes forselective analyte detection in a sample comprising a sensing body madefrom a polymeric material, preferably plastified polyvinyl chloride,comprising:

-   -   electrically conducting particles, preferably graphite powder,        which increase in concentration away from a sample contact        surface,    -   ionophore molecules, which increase in concentration towards the        sample contact surface, and    -   an electrical connection, preferably made of copper, which        passes proximal to said electrically conducting particles.

Selecting an appropriate host matrix for aptamers is one of the mainchallenges for the immobilization of aptamers in order to improve theanalytical characteristics of aptasensors. There is also a need for moresensitive and more specific potentiometric sensors, in particularelectrodes, to study molecular interactions in liquid environments witha sensorgram methodology.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide more sensitive andmore specific potentiometric sensors to determine the concentration of avariety of molecules such as vitamins, toxins, antibiotics, therapeuticdrugs, diagnostic agents, recreational drugs, catecholamines,metabolites, proteins, cells etc. in liquid environments with sensorgrammethodology and methods for using same.

Surprisingly it has been found that aptamers selected to form complexes,usually robust complexes, with target molecules such as vitamins,toxins, antibiotics, therapeutic drugs, diagnostic agents, recreationaldrugs, catecholamines, metabolites, proteins and cells, e.g. withdopamine and chloramphenicol, encapsulated in a hydrophilic gelatin Bmatrix can be used in electrochemical sensors to determine theconcentrations of such target molecules in liquid environments usingamperometric measurements or potentiometric measurements and exhibithigher sensitivity and selectivity to the target analyte than in theabsence of the gelatin B matrix. Exemplary proteins include, but are notlimited to, interferon γ. Exemplary therapeutic drugs include, but arenot limited to, cell growth factors such as vascular endothelial growthfactor, antigens (e.g. the prostate-specific antigen), promazine,lidocaine, ritodrine, bromhexine, clenbuterol, drofenine, atropine,salbutamol, trimipramine, fluphenazine, chlorpheniramine andcatecholamines such as adrenaline, dopamine and noradrenaline. Exemplarytoxins include, but are not limited to, cadaverine and dioxine,Exemplary recreational drugs include, but are not limited to, cocaineand functional equivalents thereof, and heroine.

Moreover, it has been surprisingly found that the use of type B gelatinencapsulation does not hinder the use of aptamers in such amperometricand potentiometric sensors, but also significantly increases thesensitivity of such sensors, whereas type A gelatin does not exhibitthese advantageous properties. This cannot be explained by thedifference in alkaline earth and alkali ion concentrations, sincespiking of gelatin A with comparable alkaline earth and alkali ionconcentrations had no effect on its behaviour. Without wishing to bebound by theory, this difference might be explainable by the differencein overall charge on the gelatin, since the negatively charged aptamerions, being repelled by the overall negative charge of the gelatin B,would be more mobile and hence diffuse more rapidly to the electrode ina gelatin B matrix than in a gelatin A matrix in which the overallpositive charge of the gelatin would result in attraction, and hencereduced diffusion, of the negatively charged aptamer molecules. Thisincreased diffusion also reduces the effect of impurities in thegelatin.

Just as an example, in the case of an amperometric CAP-aptasensor withan aptamer linked to a gold surface by Au—S bonding, for example, seeFIG. 1, in the absence of CAP thiolated aptamers encapsulated in gelatinB are partially un-folded and upon CAP introduction the aptamer switchesits structure to bind CAP surprisingly bringing the redox activemolecules proximate to the gold surface as characterised by theirelectrochemical behavior towards the target molecule CAP, scanningelectron microscopy (SEM), cyclic voltammetry (CV) and electrochemicalimpedance spectroscopy (EIS) measurements, see FIG. 2.

A novel method of immobilizing aptamers on electrodes using, e.g. coatedwith, ionically conductive hydrophilic matrices, preferably negativelycharged hydrophilic protein matrices like gelatin B, is provided. Apreferred example of ionically conductive matrices is gelatin Bproviding a suitable micro-environment for aptamer immobilization, whichfacilitates the electron exchange between the target molecules and theelectrode. The gelatin B film can be easily prepared and is stable overa long period. It has been found that Aptamers can be effectivelyimmobilized on gold electrode surface by incorporation within the porousnetwork of gelatin B. The three dimensional and hydrated environment ofgelatin B helps to increase the sensitivity of the developed sensor byholding the aptamer onto the electrode surface and preventing theelectrode surface from blocking. Sensors which are modified with gelatinB and aptamers show higher sensitivity toward CAP compared to thesensors without gelatin B as protective matrix. Moreover, the developedsensor is highly stable which makes it a promising technology for thefabrication of electrochemical aptasensors.

In a first aspect of the present invention, the above objective isrealised by an aptamer-based electrochemical sensor, wherein saidaptamer is covalently bonded to or chemisorbed on an electrode, saidaptamer to form a complex, usually a robust complex with a targetmolecule and is encapsulated by a gelatin B matrix.

In a second aspect of the present invention, the above objective isrealised by a method of manufacturing an aptamer-based electrochemicalsensor for determining a concentration of a target molecule comprisingthe steps of: selecting an aptamer to form a complex, preferably arobust complex with a target molecule, e.g. using the SELEX procedure;synthesizing said aptamer; adsorbing said aptamer on or covalentlycoupling said aptamer with an electrode; and providing a gelatin Bmatrix for said aptamer on said electrode thereby realising saidaptamer-based potentiometric sensor.

In a third aspect of the present invention, the above objective isrealised by the use of the aptamer-based electrochemical sensor producedaccording to the second aspect of the present invention for thequantitative electrochemical determination of a concentration of saidtarget molecule.

A specific embodiment of the present invention is the electrochemicaldetermination of a concentration of chloramphenicol by anelectrochemical sensor of the first aspect of the present invention,wherein said aptamer isAGC-AGC-ACA-GAG-GTC-AGA-TGA-CTG-AGG-GCA-CGG-ACA-GGA-GGG-CAT-GGA-GAG-ATG-GCG.

A specific embodiment of the present invention is the electrochemicaldetermination of a concentration of chloramphenicol by anelectrochemical sensor of the first aspect of the present invention,wherein said aptamer isAGC-AGC-ACA-GAG-GTC-AGA-TGA-CTG-AGG-GCA-CGG-ACA-GGA-GGG-GGA-GAG-ATG-GCG-TGA-GGT-CCT-ATG-CGT-GCT-ACC-GTG-AA.

Another specific embodiment of the present invention, the aboveobjective is realised by the use for the electrochemical determinationof a concentration of dopamine by an electrochemical sensor of the firstaspect of the present invention, wherein said aptamer isGTC-TCT-GTG-TGC-GCC-AGA-GAA-CAC-TGG-GGC-AGA-TAT-GGG-CCA-GCA-CAG-AAT-GAG-GCC-C.

A fourth aspect of the present invention relates to a compositeelectrode combining a polymeric material and electrically conductingparticles for selective analyte detection, wherein said electrode iscoated with gelatin type B. Such gelatin-coated composite electrode maybe a part of a three-electrode potentiometric cell for selective analytedetection, further comprising a reference electrode and acounter-electrode. This composite electrode and potentiometric cell maybe for an in vivo analyte sensor or an in vitro analyte sensor.

Particular and preferred aspects of the invention are set out in theaccompanying independent and dependent claims. Features from thedependent claims may be combined with features of the independent claimsand with features of other dependent claims as appropriate and notmerely as explicitly set out in the claims.

The above and other characteristics, features and advantages of thepresent invention will become apparent from the following detaileddescription, taken in conjunction with the accompanying drawings, whichillustrate, by way of example, the principles of the invention.

DEFINITIONS

The term “electrochemical biosensor”, as used herein, means ananalytical device that consist of a sensitive biological recognitionmaterial targeting an analyte of interest and a transduction element forconverting the recognition process into an amperometric orpotentiometric signal.

The term “sensorgram”, as used herein, means a plot of thepotentiometric signal vs. time, when a square concentration (block)pulse passes the sensor.

The term “sensorgram methodology”, as used herein, means the use of thesensorgram to calculate K_(d) values for the interaction betweenmolecules, e.g. using the adsorption/desorption model described in K. DeWael et al., Anal. Chemistry 84 (2012) 4921-4927.

The term electrochemical aptasensor, as used herein, means anElectrochemical sensor with at least one immobilized aptamer as asensing element.

The term aptamer, as used herein, means a synthetic oligonucleic acidsequence [single strand DNA or RNA] which can bind to a molecular targetwith high affinity and specificity, for instance due to its structuralflexibility.

The term DNA-aptamer, as used herein disclosing the present invention,means a single strand synthetic DNA sequence which has been designedspecifically to recognise and bind a particular molecular target such asa small molecule, a protein, a nucleic acid, a cell, tissue or anorganism with high affinity and specificity, for instance due to theirflexibility that results in binding to their ligands via adaptiverecognition involving conformational alteration. DNA aptamers areadvantageous over RNA aptamers due to the greater intrinsic chemicalstability of DNA.

The term ionically conductive, as used in disclosing the presentinvention, means having an ionic conductivity greater than 10⁻⁵ S cm⁻¹,with ionic conductivities greater than 10⁻⁴ S cm⁻¹ being preferred andwith ionic conductivities greater than 10⁻³ S cm⁻¹ being particularlypreferred.

The term hydrophilic, as used herein, means having an affinity forattracting, adsorbing or absorbing water.

The term hydrogel, as used herein disclosing the present invention, is acolloid in which the disperse phase (colloid) has combined with thecontinuous phase (water) to produce a viscous jellylike product.

Gelatin, as used herein, refers to a product obtained by hot waterextraction (hydrolysis) from the collagen protein from skin, bone andconnective tissues of vertebrate animals (beef, pig, horse, fish). Thereare two main types of gelatin, referred to as A- (or acid) type and B-(or limed/alkaline) type. This categorization essentially goes back tothe pre-treatment of the raw material (collagen) which will affect thecharacteristics of the gelatin extracted. Gelatin gel strength ischaracterized by the Bloom number; the higher the Bloom number, thestronger the gel.

Gelatin normally slowly swells in cold water (18° C.) and more rapidlydissolves in aqueous solutions at 40° C. and above.

The term type B gelatin or gelatin B, as used herein, means a gelatinresulting from alkaline pretreatment of collagen. Type B gelatintypically has higher calcium, potassium and sodium ion concentrationsthan gelatin A, usually in the range of 900±100 ppm, 330±50 and3600±1400 ppm respectively. Another typical difference of gelatin B isthe iso-electric point (IEP) of 4.8 to 5.2 which is almost constant andindependent of the Bloom number. This is in contrast with gelatin A,where the IEP is linked to the Bloom number and ranges from about 7 (lowBloom number i.e. 50-125) to about 9 (high Bloom number i.e. 225-325).

ABBREVIATIONS

The following abbreviations are used throughout the detailed descriptionof the present invention with the following meanings.

CV: Cyclic Voltammetry.

SWV: Square Wave Voltammetry.

CAP: chloramphenicol.

DA: dopamine.

SELEX: “selection evolution of ligands by exponential enrichment”.

MRL: Maximum residue limit.

FTIR: Fourier Transform Infrared Spectroscopy.

EIS: Electrochemical impedance spectroscopy.

SCE: Standard Calomel Electrode.

SPR: Surface Plasmon Resonance.

MES: 2-(N-morpholino)ethanesulfonic acid (a buffer).

IEP: isoelectric point.

FIA: flow injection analysis.

EDC: 1-ethyl-3(3-dimethylaminopropyl) carbodiimide.

NHS: N-hydroxysuccinimide.

SPE: screen printed electrode.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention will be described with respect to particularembodiments and with reference to certain drawings but the invention isnot limited thereto but only by the claims.

It is to be understood that although preferred embodiments, specificconstructions and configurations, as well as materials, are discussedherein for devices and methods according to the present invention,various changes or modifications in form and detail may be made, e.g.method steps or device components may be added, without departing fromthe scope and spirit of this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representation of a gelatin-containing aptasensor for CAPdetection on a gold (Au) electrode.

FIG. 2 shows cyclic voltammograms of Au (1), Gel B|APT|Au electrode (2)in blank solution and Au (3), APT|Au (4) and Gel B|APT|Au (5) electrodein 1×10⁻⁶ mol L⁻¹ of CAP solution.

FIG. 3 shows the electrochemical impedance spectra of Au (1), APT|Au (2)and Gel B|APT|Au (3) electrode in 10×10⁻³ mol L⁻¹ [Fe(CN)₆]^(4−/3−) inthe frequency range 0.1 to 100,000 Hz.

FIG. 4A and FIG. 4B show SEM images of the working surface area of agold electrode after gelatin B immobilization (a), and after aptamerincorporation (b).

FIG. 5A and FIG. 5B show in (A) the dependence of the redox peak currenton the concentration of CAP at a

-   -   Gel B|APT|Au electrode, and in (B) calibration curves obtained        at APT|Au (1) and Gel B|APT|Au (2) electrodes.

FIG. 6A, FIG. 6B, and FIG. 6C show in confocal images of hydrogels. (A):pure Gelatin B; (B):

Gelatin B+coupling agents (EDC and NHS); and (C): Gelatin B+couplingagents+fluorescent aptamer. Confocal features: pixels 7.92; red value5.58; the scale bar (white)=100 μm.

FIG. 7 shows sensorgram recordings for DA on a biosensor with a GelatinB membrane with covalently linked aptamer. Square concentration pulseswere injected for 80 s.

The concentration varied from 5×10⁻⁷ M (lower curve) to 10⁻⁶ M, 5×10⁻⁶M, 10⁻⁵ M, 5×10⁻⁵ M, and 10⁻⁴ M (upper curve).

FIG. 8 shows normalisation of the curves in FIG. 7 from time 0 to thetime corresponding to maximum response R_(max) to a 10⁻⁴ M DA injection,to show the variation with concentration.

FIG. 9 shows a Nicolskii-Eisenmann-type calibration graph. The smoothcurve is obtained from a non-linear least-squares fit to aNicolskii-Eisenmann function of the type E=E°+S, log (c_(DA)+Cst).E°=387 mV, S=60.4 mV and Cst=3.9×10⁻⁷ M.

FIG. 10 shows the differences between the aptamer-based potentiometricbiosensor (lighter upper trend line) and the negative control (darkerlower trend line, only containing a Gelatin B membrane) in respect ofthe potentiometric responses of 5×10⁻⁵M to 10⁻⁷ M DA injections in FIAafter transformation to a concentration-related signal.

FIG. 11 shows the differences between the aptamer based potentiometricbiosensor (lighter), and the negative control (darker) for injections ofDA 10⁻⁶ M on a sensor which contains an aptamer (upper curve: lighter)and on a sensor which does not contain an aptamer (lower curve: darker).

FIG. 12 shows the slope of

$\quad\begin{matrix}{d({tR})} \\{dt}\end{matrix}$versus tR (Transformed Response) curves (see equation (4) below) plottedagainst the DA concentrations which were used to record the sensorgrams.

FIG. 13 shows the manufacture of a gelatin B-containing aptasensor basedon a gold screen printed electrode (SPE) for the detection of CAP bymeans of differential pulse voltammetry.

FIG. 14 shows electrochemical impedance spectra obtained with the bareSPE of FIG. 13 (FIG. 14a ), the aptamer modified SPE (FIG. 14b ) and theaptamer/gelatin B modified SPE (FIG. 14c ).

FIG. 15 shows the differential pulse voltammograms of accumulated CAP(10⁻⁹ M) at the surface of the bare gold SPE of FIG. 13 (curve a), agelatin A modified SPE (curve b), a gelatin B modified SPE (curve c), anaptamer modified SPE (curve d), an aptamer/gelatin A modified SPE (curvee) and an aptamer/gelatin B modified SPE (curve f) in tris buffersolution.

FIG. 16A and FIG. 16B show the influence of the kind (FIG. 16A) and time(FIG. 16B) of accumulation of CAP on the electrochemical detectionsignal.

FIG. 17A and FIG. 17B show in (A) the dependence of the redox peakcurrent on the concentration of CAP at a Gel B|APT|Au SPE electrode, andin (B) calibration curves obtained at APT|Au SPE (1) and Gel B|APT|AuSPE (2) electrodes.

ELECTRODE

The electrochemical aptasensor according to the present inventionrequires at least a detecting electrode, also named a working electrode.A conventional electrochemical device is a three-electrode cellconfiguration comprising a reference electrode (such as, but not limitedto, a calomel electrode or a silver electrode) and a counter-electrode.Suitable detecting electrodes include gold electrodes, glassy carbonelectrodes, an inert metal in an ionically conducting composite, andcomposite electrodes combining a polymeric material and electricallyconducting particles. The electrode may be obtained by any manufacturingprocess known in the art, including the screen printing technique formaking a SPE.

WO 2005/103664, the content of which is incorporated herein byreference, discloses suitable composite potentiometric electrodes forselective analyte detection-according to the present invention, providedthat said electrodes are coated with an ionically conductivehydrophilic, preferably negatively charged, matrix such as gelatin typeB or an equivalent thereof, for instance in the form of a thin (μmscale) or ultrathin (nm scale) layer. Gelatin B for performing thisaspect of the invention may have any Bloom number, including the lowrange 50-125, the medium range 125-225 and the high range 225-325.

This composite electrode, and a potentiometric cell including it, may befor an in vivo analyte sensor or an in vitro analyte sensor.

Aptamer-Based Electrochemical Sensor

In a first aspect of the present invention, the above objective isrealised by an aptamer-based electrochemical sensor, wherein saidaptamer is covalently bonded to or chemisorbed on an electrode, saidaptamer is selected to form a complex, usually a robust complex with atarget molecule and is encapsulated by an ionically conductivehydrophilic, preferably negatively charged, matrix such as gelatin B oran equivalent thereof. Gelatin B for performing this aspect of theinvention may have any Bloom number, including the low range 50-125, themedium range 125-225 and the high range 225-325.

According to a preferred embodiment of the first aspect of the presentinvention, said aptamer is selected with the SELEX procedure, or it maybe known from the literature.

According to another preferred embodiment of the first aspect of thepresent invention, said target molecule is selected from the groupconsisting of vitamins, antibiotics, toxins, therapeutic drugs,diagnostic agents, recreational drugs (e.g. cocaine), catecholamines,metabolites, proteins and cells.

According to another preferred embodiment of the first aspect of thepresent invention, said aptamer is5′-SH—(CH2)6-AGC-AGC-ACA-GAG-GTC-AGA-TGA-CTG-AGG-GCA-CGG-ACA-GGA-GGG-CAT-GGA-GAG-ATG-GCG-3′and is intended for CAP detection.

According to another preferred embodiment of the first aspect of thepresent invention, said aptamer is5′-GTC-TCT-GTG-TGC-GCC-AGA-GAA-CAC-TGG-GGC-AGA-TAT-GGG-CCA-GCA-CAG-AAT-GAG-GCC-C-spacer-NH₂-3′and is intended for dopamine detection.

Method of Manufacturing an Electrochemical Sensor

In a second aspect of the present invention, the above objective isrealised by a method of manufacturing an aptamer-based electrochemicalsensor for determining a concentration of a target molecule comprisingthe steps of:

-   -   selecting an aptamer to form a complex, preferably a robust        complex, with a target molecule, e.g. using the SELEX procedure;    -   synthesizing said aptamer;    -   adsorbing said aptamer on or covalently coupling said aptamer        with an electrode; and    -   providing an ionically conductive hydrophilic, preferably        negatively charged, matrix, preferably gelatin type B or an        equivalent thereof, for said aptamer on said electrode.

According to a preferred embodiment of the second aspect of the presentinvention, said target molecule is selected from the group consisting ofvitamins, antibiotics, toxins, therapeutic drugs, diagnostic agents,recreational drugs, catecholamines, metabolites, proteins and cells.

Use of the Aptamer-Based Electrochemical Sensor

In a third aspect of the present invention, the above objective isrealised by the use of the aptamer-based electrochemical sensoraccording to the first aspect of the present invention or producedaccording to the second aspect of the present invention for theelectrochemical determination of a concentration of a target molecule.

According to a preferred embodiment of the third aspect of the presentinvention, said electrochemical determination is a potentiometricdetermination.

According to another preferred embodiment of the third aspect of thepresent invention, said electrochemical determination is an amperometricdetermination.

EXAMPLES

Chemicals and Materials

Promazine, lidocaine, ritodrine and chloramphenicol (CAP) were obtainedfrom Sigma-Aldrich (Bornem, Belgium) and dopamine (DA) was obtained fromFluka. To dissolve these drugs, small amounts of ethanol (Fluka,analytical grade) were used.

MES was obtained from Acros Organics (Eupen, Belgium).

The coupling agents EDC and NHS were obtained from Sigma-Aldrich.

All chemicals were of analytical reagent grade.

Type B gelatin (Gel, IEP=5, Bloom strength=257), isolated from bovineskin by the alkaline process, was provided by Tessenderlo Chemic(Belgium).

Buffers:

Tris buffer containing:

NaCl 100 × 10⁻³ mol L⁻¹  Tris HCl 20 × 10⁻³ mol L⁻¹  MgCl₂ 2 × 10⁻³ molL⁻¹ KCl 5 × 10⁻³ mol L⁻¹ CaCl₂ 1 × 10⁻³ mol L⁻¹with a pH of 7.6 obtained from VWR (Belgium) and used as a bindingbuffer solution.Aptamers:

binding aptamer sequence from Eurogentec. (5′-SH-(CH2)₆-AGC-AGC-ACA-GAG-GTC-AGA-TGA-CTG- AGG-GCA-CGG-ACA-GGA-GGG-CAT-GGA-GAG-ATG-GCG-3′) A 58 mer aptamer selected  aptamer synthesized specifically to detect DA by Integrated DNA (Zheng et al., 2011)Technologies (5′-GTC-TCT-GTG-TGC-GCC- (Leuven, Belgium)AGA-GAA-CAC-TGG-GGC-AGA- TAT-GGG-CCA-GCA-CAG-AAT-GAG-GCC-C-spacer-NH₂-3′)Voltammetric Measurements

Voltammetric measurements were recorded by μ-AutolabII potentiostatcontrolled by NOVA 1.7 software package (Metrohm, The Netherlands).Electrochemical impedance spectroscopy (EIS) measurements were performedby using a frequency response analyzer module. A gold electrode inlaiddisk (Φ=1.6 mm) was used as working electrode. A saturated calomelelectrode (SCE) and graphite were used as the reference and theauxiliary electrode, respectively.

Morphological Investigation

Morphological investigation of the electrode surface was performed usinga JEOL JSM-6300 Scanning Electron Microscope (SEM) and confocalmicroscopy was performed by localizing Cy3 dyes with a Nikon C1 laserscanning confocal unit (D-eclipse-C1, Nikon, Melville, N.Y.) equippedwith an argon and a helium/neon laser line fitted onto an uprightmicroscope (Eclipse E600, Nikon, Melville, N.Y.) in combination with a10× planfluor (NA: 0.50) objective manufactured by Nikon (Melville,N.Y.).

FIA

The FIA recordings were performed using a LC-10ADvp pump (ShimadzuLiquid Chromatography) and a Rheodyne 7125 six port external sampleinjector (VICI, US). A 1.00 mL sample loop was used to generate squareconcentration pulses for sensorgram recording in FIA conditions. ThePEEK tubing (Alltech, USA) of the injection loop and theinjector-detector connections had an internal diameter of 0.18 mm. Theflow rate was 1.00 mL min⁻¹. Poiseuille peak broadening effects werekept to a minimum using short injector-detector connections (150 mm). Toavoid such effects at the end of the square concentration pulse, theinjector was switched from inject to load after 80 s (well before thesample loop volume was totally emptied).

This results in a sharp pulse with negligible broadening as well at thestart as at the end of the pulse. The eluent was 10.0 mM MES. pH 7.0.

The column outlet was directed perpendicularly towards the sensitivemembrane of the coated wire electrode in a “wall-jet” flow cell. Thedistance from the LC tubing outlet to the electrode was 0.10 mm.

Preparation of the Amperometric Electrode for Example 1

Prior to surface modification, the gold electrode was mechanicallypolished with 1.0 and 0.05 μm alumina slurry separately, followed byrinsing thoroughly with double distilled water. Then it was washedultrasonically in double distilled water then ethanol for 15 minutes.The electrode was rinsed with distilled water and dried at roomtemperature. Subsequently, electrodes were electrochemically cleaned bypotential scanning between −1.2 and 1.2 V until a reproducible cyclicvoltammetric scan was obtained.

To immobilize the CAP-binding thiolated aptamer on the gold surface,3×10⁻⁶ L of a 2.5×10⁻⁶ mol L⁻¹ aptamer and 5% (w/w) gelatin type Bsolution was dropped onto a freshly smoothed gold surface, and thesolvent was then evaporated at 4° C. for 6 hours. The final sensinginterface was ready after rinsing with buffer solution.

Preparation of the Potentiometric Electrode for Example 2

The indicator electrode is made of a PVC cylinder. It contains acylindrical substrate electrode (3.0 mm diameter×1.0 mm length), whichis an electronically conducting graphite/PVC composite material. Thecomposite substrate electrode was polished with Carbimet grid 600(Buehler Ltd, USA).

To coat the gelatinous hydrogel on the electrode, 10.0 μL of a mixture,which consists of 25.0 mg gelatin B dissolved in 0.50 mL 10.0 mM MES (5%w/v) pH 7.0 at 40° C., was brought onto the electrode surface with amicropipette and exposed to air for 2 hours at 4° C. (drop drying).

The EDC-NHS coupling procedure was used to bind the DA-binding aminatedaptamer covalently to the gelatin B hydrogel (which contains carboxylgroups). After adding 20 μL of the coupling agents (15.32 mg of EDC and2.32 mg of NHS dissolved in 100 μl of 10.0 mM MES buffer at pH 7.0) tothe coated hydrogel for 2 hours, 2×4 μL of the aptamer (10⁻⁴M) wasapplied for 1 hour. After evaporation for 2 hours at 4° C. theelectrodes were kept in 10.0 mM MES running buffer, pH 7.0 for at least3 hours until a stable baseline was obtained. The sensorgrams of eachanalyte were measured on 3 electrodes, and used when the inter-electrodereproducibility was better than 10%. At least 3 sensorgrams (injections)were recorded on each electrode after conditioning and stabilization inthe running buffer.

The membrane potential was measured against an Orion 800500Ross®reference electrode (Ag/AgCl) using a high impedance (10¹³Ω) homemadeamplifier. The detection signals were recorded on a data stationcomposed of a computer equipped with a 6013 NI DA converter and LabVIEW7 (National Instruments, US) based software. The overall RC timeconstant of the high impedance amplifier plus data station was set to0.2 s.

Example 1 Electrochemical CAP-aptasensor

Morphology

The morphology of the prepared electrodes was investigated by SEMmeasurements (see FIG. 4). The SEM image of a gelatin assembled goldelectrode is shown in FIG. 4(a), a regular and porous structure beingobserved. The gelatin film was compact, smooth and homogenous withoutgrainy and porous structure, showing that an ordered matrix was formed.However, a rough, sponge-like and irregular surface appeared whenaptamers were added to the matrix, indicating a clear morphology changedue to the interactions.

Electrochemical Behaviour:

Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS)measurements were performed to characterize an APT|Au electrode and aGel B|APT|Au electrode as shown in FIGS. 2 and 3 respectively.

Whereas at a bare gold electrode no obvious redox peak was observed inthe CV-characteristic (indicated by 3 in FIG. 2), a large reduction peakwas observed in the CV-characteristic (indicated by 4 in FIG. 2) atAPT|Au. With gelatin type B encapsulation the response (indicated by 5in FIG. 2) increased due to the increased electron transfer capabilityof the modified electrode. A gelatin type B film acts as both asupporting polymer for aptamers and as a reaction medium. Therefore,substrate mass transport occurred in and out of the hydrogel layer whilethe aptamers were retained in the matrix and on the electrode surface.

The EIS characteristics shown in FIG. 3 demonstrate that the electrontransfer resistance increased in the following order: Au electrode(indicated by 1 in FIG. 3), APT|Au electrode (indicated by 2 in FIG. 3)and Gel B|APT|Au electrode (indicated by 3 in FIG. 3). This isattributed to the fact that ssDNA with negative charges on its phosphatebackbone makes an electrostatic repulsive force to the[Fe—(CN)₆]^(3−/4−) anions and prevents electrons from reaching theelectrode surface. The increase in electron transfer resistanceindicates that the aptamers are successfully immobilized on theelectrode surface. In the case of a Gel B|APT|Au electrode, anadditional barrier of negatively charged gelatin has increased theelectron transfer resistance which results in a larger semi-circle. Thisdemonstrates the successful immobilization of APT on the electrode andthe blocking effect of the gelatin layer to unspecific redox activemolecules. The CV-characteristics of the resulting aptasensor atdifferent scan rates were also studied. In the presence of CAPmolecules, a linear dependence of peak current upon scan rate impliedthat the electrochemical process was a surface confined process. Theshort-term stability of the aptasensor was investigated over successivecyclic voltammograms. The response current of the electrode withimmobilized aptamer and gelatin encapsulation decreased by about 3.2% ofits initial response and the relative standard deviation (R.S.D.) was1.8%, whereas the electrode only with immobilized aptamer showed a 20.2%decrease and an R.S.D. of 3.5%. The high stability could be attributedto the high chemical stability of gelatin and aptamers, both improvingthe stability of the aptasensor. The good stability of the Gel B|APT|Aucan be ascribed to excellent biocompatibility and the stabilizingmicroenvironment around the aptamers provided by the modified gelatinsensors.

From FIG. 5(A), it can be seen that the peak current increases withincreasing CAP concentration. There was a linear relationship betweenpeak current and CAP concen-tration in the range from 2.1×10⁻⁹ to5.2×10⁻⁷ mol L⁻¹. The linear regression equation was I=−1.6 C+2.7,(units for C and I are ×10⁻⁹ M and ×10⁻⁷ A respectively) and thecorrelation coefficient is 0.9684. The detection limit was 1.2×10⁻⁹ molL⁻¹ based on interpolation to the point where the current differs fromthe background current for three standard deviations calculated from thecurrents obtained for three different electrodes. In the case of APT|Auelectrodes (without gelatin as a protective matrix), the detection limitwas found to be 1.6×10⁻⁹ and the linear range was between 1.6×10⁻¹ and4.2×10⁻⁷ mol L⁻¹ which demonstrated the improved performances of the GelB|APT|Au electrode over the APT|Au electrode. The enhanced sensitivityand broader dynamic range could be due to the ability of gelatin B tomake diffusion of analytes easier and prevent aptamers from leaking andmost important, prevent the electrode surface from undesired adsorption.A comparison of the calibration curves for the two electrodes [see FIG.5(B)] shows that the sensitivity (slope) of the Gel B|APT|Au electrodewas higher than that of the APT|Au electrode.

The assay of this target in real samples was investigated by detectingCAP in a sample of skimmed cow's milk. The standard addition method wasemployed to evaluate the applicability of the developed aptasensor. Theincreased reduction peak of CAP compared with the reduction currentobtained at an aptamer immobilized electrode without gelatin asprotective matrix occurred at a Gel B|APT|Au electrode in the expectedpotential range, which indicated an enhanced sensitivity of thedeveloped Gel B|APT|Au electrode. Recovery values between 87% and 94%were obtained, indicating the applicability of the developed aptasensorfor CAP detection in real biological samples.

Example 2 Electrochemical DA-aptasensor

Morphology

The covalent binding of the aminated oligonucleotides to the gelatin B,which contains carboxyl groups, was examined by confocal microscopy. Toexclude the background signal, pure Gelatin B and Gelatin B treated withcoupling agents (EDC and NHS), were checked as blanks. No signal wasobserved in the latter cases (FIGS. 6A and 6B). This is in contrast withthe bright fluorescent signal observed for a hydrogel treated with afluorescent labeled (Cy3) aminated aptamer (FIG. 6C). Even after threehours of use in the FIA potentiometric set-up, the intense fluorescencepersisted. This experiment confirmed the covalent coupling of theaminated oligonucleotides to the gelatin B.

Electrochemical Behaviour

After checking the coupling of the aptamer to the biopolymer asdescribed above, the electrodes were placed in a FIA potentiometricsetup. After injection of an analyte in the FIA system, the voltageoutput varies with time, due to the kinetics of complex formationbetween DA and the anti-DA aptamer. A positively charged analytemolecule such as DA has the tendency to adsorb to the sensor coating, ifa DA binding aptamer is present in this material. This provokes asurface potential change, which is our analytical signal. In its mostsimple representation, the eluent/gelatin B interface is expected tohave a Boltzmann type distribution of positive charges as the gelatin Bbehaves as a cation exchange-like material in the given conditions.

The target molecules were injected as square concentration pulses,comparable with the block pulses in SPR. The sensorgrams (mV responsesas a function of time) obtained are shown in FIG. 7. Using ahydrodynamic method and square concentration pulses has the advantagethat both adsorption as desorption curves can be obtained and theadsorption/desorption kinetics calculated.

FIG. 7 shows the potentiometric response of DA on the sensor whichcontains the DA specific aptamer. The response heights (mV obtained atthe plateau values after 80 s of injection) are concentration dependent:see FIGS. 7 and 9. After normalization of the curves from time zero toR_(max) (see FIG. 8), it is clear that higher concentrations of DA showfaster “on” kinetics than lower concentrations. This concentrationdependent difference in adsorption kinetics (rising part of the curve)is typical for SPR experiments.

If the maximum responses (in mV) are plotted against the logarithm ofthe concentration, the typical Nicolskii-Eisenmann curve is obtained(see FIG. 9). At higher concentration values (results not shown),saturation of the signal (reaching a plateau value) starts to occur.This is ascribed to the fact that a relatively low aptamer concentration(10⁻⁴ M) was used to bind to the hydrogel. DA concentrations below5×10⁻⁵ M were therefore used in these experiments.

The Nicolskii-Eisenmann equation (see FIG. 9) was transformed toequation 1, to obtain a concentration dependent output signal(“Transformed Response”, tR) of the sensor.tR=(10^(mV/S)−1)·Cst  (1)

The transformed R_(max) values (equation 1) of the potentiometricsensors with coupled aptamer were compared with the values obtained withelectrodes which did not have an aptamer coupled to the hydrogel: seeFIG. 9. Four electrodes were tested, each with 3 injections. From fiveto ten times higher signals were measured with the aptamer-containingelectrodes. Not only the height, but also the shape of the sensorgramwas different when comparing gelatin B membranes with and withoutcoupled aptamer (see FIG. 9). The gelatin B electrodes containingaptamer always yielded slowly falling curves. This is additionalevidence for the recognition by the aptamer of the target molecule.Slowly falling curves mean slow desorption kinetics. This yields smallk_(off) values, resulting in high K_(association) values: see equation.2.

$\begin{matrix}{K_{association} = \frac{k_{on}}{k_{off}}} & (2)\end{matrix}$

FIGS. 9, 10 and 11 clearly show that the aptamer-based sensors are verysensitive, the responses being are at least a factor of 800 higher (on amV basis) than those of the classical PVC-based electrodes.

Determination of the Association Constant, K_(ass), Between DA-SpecificAptamer and DA

The above-defined “sensorgram methodology” was used. Sensorgrams wererecorded at different analyte concentrations. The rising parts of thesensorgrams are shown in mV in FIG. 8. The mV y-axis in thesesensorgrams was first converted to a concentration dependent response,called transformed Response (see equation. 1). The transformed Response(tR) of the sensor was linearly related to the number of occupiedadsorption sites, R_(occupied), i.e.: transformed Response(tR)˜R_(occupied).

The rate of adsorption of DA, v_(on), can be regarded as a reaction ratewhich is first order in the DA concentration in the bulk of the solution(c_(analyte)) and in the concentration of free adsorption sites (oraptamers) on the sensor surface: R_(max)−R_(occupied). It can bedescribed by a rate equation of the form of equation 3:

$\begin{matrix}{v_{on} = {\frac{{dR}_{occupied}}{dt} = {{k_{on} \cdot {c_{analyte}\left( {R_{\max} - R_{occupied}} \right)}} - {k_{off}R_{occupied}}}}} & (3)\end{matrix}$

This equation can be rewritten by substituting the concentration of DAmolecules, which adsorbed onto (or “occupied”) the aptamer derivatizedsurface. R_(occupied), by the sensor's transformed Response, tR, and byreplacing R_(max) by tR_(max). This yields equation 4:

$\begin{matrix}{\frac{d({tR})}{dt} = {{k_{on} \cdot c_{analyte} \cdot {d({tR})}_{\max}} - {\left( {{k_{on} \cdot c_{analyte}} + k_{off}} \right) \cdot {tR}}}} & (4)\end{matrix}$The first derivative of the rising up-going part of the transformedResponse (tR) with time

$\frac{d({tR})}{dt},$was plotted against the transformed Response, tR. This yielded astraight line with a slope equal to −(k_(on)·c_(analyte)+k_(off)).Plotting this slope versus c_(analyte) for the set of analyteconcentrations (DA) yielded a graph from which k_(on) and k_(off) werecalculated. The “Slope” versus DA concentration curve obtained is givenin FIG. 12, which confirms that the model used for the sensorgrammethodology is applicable. For the first time reliable molecularinteraction data were obtained for the interaction of a biomolecule (theaptamer) with its target molecule, via a potentiometric sensorprinciple.

Specific Detection with DA-Specific Aptamer

To check the specificity of the DA-specific aptamer, Dopamine and threeother basic drugs (ritodrine, lidocaine and promazine) were tested onthe gelatin B-coated electrodes with and without the aptamer. Theselipophilic cationic drugs have much better responses on PVC-basedpotentiometric sensors than DA. Sensors based on gelatin B were quiteinsensitive towards these three other drugs, there being no improvementin sensitivity for these compounds when the aptamer biorecognitionelement was coupled to the gelatin B, whereas DA showed a very clearincrease in R_(max), as disclosed above. Table 1 gives the R_(max)values (in mV) of 10⁻⁵ M injections of different analyte molecules fordifferent potentiometric electrodes.

TABLE 1 Dopamine Ritodrine Lidocaine Promazine response in response inresponse in response in Coating mV mV mV mV Gelatin B 9.46 1.83 2.883.66 Gelatin B + 92.86 1.57 1.57 2.44 DA-specific aptamer

Example 3 Electrochemical CAP-aptasensor Based on a SPE Gold Electrode

Electrochemical measurements were recorded by a Autolab potentiostatcontrolled by NOVA 1.10 software package (Metrohm, The Netherlands).Morphological investigation of the electrode surface was done on a FeiQuanta 250 FEG Scanning Electron Microscope (SEMI). The SPE waspurchased from Metrohm and made of a gold working electrode, a carboncounter electrode and a silver reference electrode.

Unless specified otherwise, the chemicals and materials are the same asin the previous examples.

FIG. 13 schematically shows the construction of the electrochemicalgelatin type B-based CAP-aptasensor. In the absence of CAP, thethiolated aptamer encapsulated in gelatin B is partially un-folded butlinked to the gold surface by an Au—S bonding. When CAP is introduced tothe modified SPE, the aptamer switches its structure to bind CAPbringing the redox active molecules proximate to the electrode surfaceresulting in an enhanced electron transfer.

The first step is the electrochemical pre-treatment and biomodificationof the gold screen printed electrode (SPE). Prior to immobilization ofthe thiolated DNA aptamer, a multiple-pulse amperometric pre-treatmentof the gold surface is carried out in a stirred 0.5 mol L⁻¹ H₂SO₄, 10mmol L⁻¹ KCl solution. The following triple-potential pulse sequence:−0.3 V for 3.0 s; 0.0 V for 3.0 s and +1.0 V for 1.5 s (15 cycles) wasapplied. The gold working electrode surface of SPE was then exposed tothe mixture of aptamer (5 μM) and the solution of gelatin type B (5 w/v%) in tris buffer (pH 7.6). The percentage of the incorporation was70:30 v/v % from the aptamer:gel mixture. Chemisorption is allowed toproceed (about 4 hours) while the electrodes are stored in a wet chamberto protect the solution from evaporation. The immobilization step isfollowed by addition of CAP solution (a 100 μL drop) on top of themodified-gold SPE for 25 minutes. Prior to the electrochemicalmeasurement, the electrode was gently washed with 100 μL of tris buffer.Then, the differential pulse voltammetry is performed in tris buffersolution (pH 7.6).

EIS measured data (FIG. 14) show that the electron transfer resistanceincreases in the following order: bare SPE (FIG. 14a ), aptamer modifiedSPE (FIG. 14b ) and aptamer/Gelatin B modified SPE (FIG. 14c ). Theincrease in electron transfer resistance indicates that the aptamers aresuccessfully immobilized on the electrode surface. In the case of aaptamer/GelB SPE, an additional barrier of negatively charged gelatinhas increased the electron transfer resistance which results in a largersemi-circle. This demonstrates the successful immobilization of theaptamer on the SPE electrode and the blocking effect of the gelatin Blayer against unspecific redox active molecules.

In order to investigate the role of the gelatin B matrix in theefficiency of the aptasensor, differential pulse voltammetry (DPV) wasselected as sensitive technique. FIG. 15 displays the differential pulsevoltammograms of accumulated CAP (10⁻⁹ M) at the surface of bare goldSPE (curve a: 0.00 μA), Gelatin A modified SPE (curve b: 0.67 μA±0.02),Gelatin B modified SPE (curve c: 0.801±0.015 μA), aptamer modified SPE(curve d: 1.22±0.06 μA), aptamer/Gelatin A modified SPE (curve e:0.86±0.04 μA) and aptamer/Gelatin B modified SPE (curve f: 2.71±0.09 μA)in tris buffer solution. The indicated currents reflect the peak currentobtained in the voltammogram and can be explained as the irreversiblereduction of the nitro group (NO₂) present in CAP molecules, withformation of hydroxylamine (NHOH). As can be seen, there is no signalfor CAP at a bare gold electrode. It means that no chemisorption takesplace after 25 minutes accumulation of CAP at the surface of bare goldSPE (curve a). After immobilization of gel at SPE, the DPV signalappears for accumulated CAP (curve b and c). However, Gelatin B modifiedSPE shows a higher current signal than Gelatin A. Following themodification of the SPE by the aptamer, the current of DPV signalincreased (curve d), confirming the ability of the synthesized aptamerto capture the target molecule. The combination of aptamer and Gelatin Ahad no positive effect on the sensor efficiency (curve e) while theincorporation of aptamer and Gelatin B showed a very good responsetoward the target molecule (curve f) at the potential at which we expectthe reduction of the nitro group, i.e. −0.7 V. Also, there is a preoxidation wave around −0.45V that shows the presence of intermediates inredox reactions of CAP.

Dramatic increase in DPV height after mixing aptamer and Gelatin B islikely due to an increase in charge transfer kinetics resulting from thebetter reactivity of the aptamer towards the target. Due to theincorporation of the aptamer in the gelatin B matrix and itsbiocompatibility, most sites of the aptamer will remain active duringthe formation of the self-assembled monolayer from the thiolatedaptamer. As the mixture of aptamer/gelatin B shows a significantlyhigher DPV signal than aptamer/gelatin A, the biocompatibility ofgelatin B towards aptamers is expected to be better. Because of thephysical interactions between the aptamer chains and gelatin (e.g. vander Waals forces and hydrogen bonds between amino acids), GelB is a goodexample of a physically cross-linked hydrogel. Therefore, thehydrophilic groups or domains which are hydrated make GelB a suitablematrix for the entrapment of the aptamer.

To obtain the most sensitive results, parameters such as the kind (FIG.16A) and time (FIG. 16B) of the CAP accumulation step were optimized forthe electrochemical detection of CAP. Three approaches were investigatedto accumulate CAP on the surface of the aptamer/GelB electrodes. First,the modified SPE was immersed in the CAP solution while the solution wasstirred very fast (i). Secondly, the CAP solution was stirred slowly(ii). Thirdly the modified SPE was kept horizontally with a 100 μL ofthe CAP solution on top of it (wet drop, iii). The latter was proven tobe the best to accumulate CAP because of the better interactionopportunity between CAP, aptamer, GelB and the electrode surface (FIG.16A). Also, the time of accumulation for CAP on the surface of theaptamer/GelB modified SPE was investigated during 10, 15, 20, 25, 30 and35 minutes. When accumulation time increased from 10 to 25 minutes, thesignal of the accumulated CAP enhanced. After that, no obvious changesin the DPV reduction current of CAP were observed. Therefore, 25 minuteswas selected as optimized time (FIG. 16B).

FIG. 17A depicts the diagnostic performance of the aptasensor. To studythe role of gelatin B as matrix in function of the biosensor, the CAPreduction signal was investigated on the aptamer/gelatin B modified SPE.It can be seen that the peak current increases with an increase of theconcentration of CAP (FIG. 17B). The peak current shows a linearrelationship with the Log of concentration of CAP in the range from 0.10to 10.0 pico mol L⁻¹ (insert of FIG. 17B). The calculated detectionlimit is 2.09×10⁻¹⁴ mol L⁻¹ based on the interpolation to the pointwhere the current differs from the background current for three standarddeviations calculated from the currents obtained for three differentelectrodes.

Example 4 Selective CAP Detection in a Milk Sample

The assay of the target in a real sample was investigated by detectingCAP in a skimmed cow's milk sample with the SPE electrode of example 3.The standard addition method was employed to evaluate the applicabilityof the developed aptasensor. The increased reduction peak of CAPoccurred in the expected potential range at aptamer/gelatin B modifiedSPE compared to the reduction current obtained at an aptamer immobilizedelectrode without a gelatin protective matrix, suggesting an enhancedsensitivity of the developed sensor. Recovery values shown in table 2,ranging between 82% and 95%, indicate the applicability of the developedaptasensor for CAP detection in real samples.

TABLE 2 Added Recovery R.S.D. Sample (M) Detected (M) (%) (%) 1 10⁻⁹9.50 ± (0.36) × 95 3.78 10⁻¹⁰ 2 10⁻¹⁰ 8.60 ± (0.30) × 86 3.48 10⁻¹¹ 310⁻¹¹ 8.23 ± (0.26) × 82 3.15 10⁻¹²

For selectivity study, thiamphenicol and florfenicol antibiotics with astructure similar to CAP were used. Results showed that they did notinfluence the performance of the aptasensor, suggesting a goodselectivity of this aptasensor. Also for stability study, the impedancemeasurement was done after a CAP electrochemical detection. The samevalue was obtained, indicating that the self-assembled aptamer is quitestable.

The invention claimed is:
 1. A composite electrode combining a polymericmaterial and electrically conducting particles, wherein said electrodeis coated with a type B gelatin.
 2. A composite electrode according toclaim 1, wherein the polymeric material is plasticized polyvinylchloride.
 3. A composite electrode according to claim 1, wherein theelectrically conducting particles are graphite powder.
 4. A compositeelectrode according to claim 1, wherein the polymeric material isplasticized polyvinyl chloride and wherein the electrically conductingparticles are graphite powder.
 5. A composite electrode according toclaim 1, wherein an aptamer is covalently bonded to, or chemisorbed on,said electrode.
 6. A composite electrode according to claim 1, whereinan aptamer is covalently bonded to, or chemisorbed on, said electrode,wherein the aptamer is selected to form a robust complex with a targetmolecule, and wherein the target molecule is selected from the groupconsisting of vitamins, toxins, antibiotics, therapeutic drugs,diagnostic agents, recreational drugs, catecholamines, metabolites,proteins, and cells.
 7. A composite electrode according to claim 1,wherein an aptamer is covalently bonded to, or chemisorbed on, saidelectrode, wherein the aptamer is selected to form a robust complex witha target molecule, and wherein the target molecule is dopamine and theaptamer isGTC-TCT-GTG-TGC-GCC-AGA-GAA-CAC-TGG-GGC-AGA-TAT-GGG-CCA-GCA-CAG-AAT-GAG-GCC-C(SEQ ID NO:3).
 8. A composite electrode according to claim 1, wherein anaptamer is covalently bonded to, or chemisorbed on, said electrode,wherein the aptamer is selected to form a robust complex with a targetmolecule, and wherein the target molecule is chloramphenicol and theaptamer isAGC-AGC-ACA-GAG-GTC-AGA-TGA-CTG-AGG-GCA-CGG-ACA-GGA-GGG-CAT-GGA-GAG-ATG-GCG(SEQ ID NO:1) or the aptamer isAGC-AGC-ACA-GAG-GTC-AGA-TGA-CTG-AGG-GCA-CGG-ACA-GGA-GGG-GGA-GAG-ATG-GCG-TGA-GGT-CCT-ATG-CGT-GCT-ACC-GTG-AA(SEQ ID NO:2).
 9. A composite electrode according to claim 1, wherein anaptamer is covalently bonded to, or chemisorbed on, said electrode,wherein the aptamer is selected to form a robust complex with a targetmolecule, and wherein the target molecule is cocaine or heroin.
 10. Acomposite electrode according to claim 3, wherein an aptamer iscovalently bonded to, or chemisorbed on, said electrode.
 11. A compositeelectrode according to claim 3, wherein an aptamer is covalently bondedto, or chemisorbed on, said electrode, wherein the aptamer is selectedto form a robust complex with a target molecule, and wherein the targetmolecule is selected from the group consisting of vitamins, toxins,antibiotics, therapeutic drugs, diagnostic agents, recreational drugs,catecholamines, metabolites, proteins and cells.